Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

Asexual fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> is potentially heterothallic

Verticillium dahliae, a soilborne plant pathogen, causes wilt disease in many important crops. We reported previously that the mating type gene MAT1-2-1 is spread to isolates of this asexual fungus. However, we did not determine whether V. dahliae is homothallic or heterothallic because the opposite mating type gene, MAT1-1-1, had not been identified. In the present study, we identified the MAT1-1-1 gene from an isolate lacking MAT1-2-1 and the mating type idiomorphs of V. dahliae. Each isolate we tested contained either the MAT1-1 or MAT1-2 idiomorph, indicating that the asexual fungus V. dahliae is potentially heterothallic.     

Involvement of two different types of Verticillium dahliae in lettuce wilt in Ibaraki Prefecture, Japan

Although wilt diseases of various plants by Verticillium dahliae occur worldwide, Verticillium wilt of lettuce is relatively new and rare. In 2009, this disease appeared suddenly in commercial fields in Ibaraki Prefecture, Japan. To investigate the cause of the disease, we characterized each lettuce isolate for pathogenicity, mating types, and genetic characteristics and found two types of V. dahliae. A population that included at least two types of V. dahliae was the putative infection source of the disease. Effective management systems against V. dahliae isolates of different races and having different host ranges are necessary.     

Characterization of new monoclonal antibodies against porcine lymphocytes: molecular characterization of clone 7G3, an antibody reactive with the constant region of the T-cell receptor delta-chains

A battery of mouse monoclonal antibodies (mAbs) reactive with porcine peripheral blood (PB) leukocytes was generated. Among the mAbs, 6F10 was found to react probably with cluster of differentiation (CD)8 alpha-chain, while 7G3 and 3E12 were found to recognize gammadelta T-cells, as revealed by two-color flow cytometric and immunoprecipitation studies. 7G3 was shown to react with the constant (C) region of the T-cell receptor (TCR) delta-chain by the following facts: (1) 7G3 immunoprecipitated full-length TCR delta-chain protein fused with glutathione S-transferase (GST) produced by Esherichia coli and (2) 7G3 reacted with TCR delta-chain expressing Cos-7 cells transfected with either full-length or N-terminal deleted mutant cDNA, but did not react with Cos-7 cells transfected with C-terminal deleted mutant TCR delta-chain cDNA. All three mAbs produced high-quality immunostaining results on frozen sections, revealing a distinct distribution of gammadelta T-cells and CD8(+) cells. This report precisely characterizes mAbs against porcine TCR for the first time, facilitating molecular biological investigations of the porcine immune system.     

Retrotransposon-like elements in the genome of Verticillium dahliae may be used as DNA markers for fungal species and pathotypes

Using differential hybridization, two DNA fragments, VDf35 and VDf90, specific to Verticillium dahliae, were isolated. These fragments contained truncated open reading frames (ORFs) homologous to the gypsy-type retrotransposon. The ORFs of VDf35 and VDf90 were pol and gag homologs, respectively. In addition, VDf90 had a pol homolog without an ORF sequence. The pol homologs in VDf35 and VDf90 were similar to each other, and these two DNA fragments had completely identical sequences. Genomic Southern analysis revealed that numerous copies of these homologs existed in V. dahliae, suggesting that V. dahliae carries a gypsy-like retroelement. Genomic Southern and polymerase chain reaction (PCR) analysis also indicated that a large number of these homologs exist in V. longisporum as well as in V. dahliae, but only a few were present in V. albo-atrum. No homolog was found in either V. nigrescens or V. tricorpus. The uneven distribution of these homologs of the retroposon-like elements among Verticillium species suggested a close genetic kinship between V. dahliae and V. longisporum. PCR primers designed from VDf35 showed species- or pathotype-specific amplification. Therefore, this sequence may be useful as a DNA marker to identify species and pathotypes of V. dahliae. This is the first report on a retrotransposon-like sequence in the genome of phytopathogenic Verticillium species.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB095264 (VDf90) and AB095265 (VDf35)     

Cerebroside elicitor confers resistance to fusarium disease in various plant species

ABSTRACT In the rice blast fungus pathosystem, cerebroside, a compound categorized as a sphingolipid, was found in our previous study to be a non-racespecific elicitor, which elicits defense responses in rice. Here we describe that cerebroside C is produced in diverse strains of Fusarium oxysporum, a common soilborne agent of wilt disease affecting a wide range of plant species. In addition, some type of cerebroside elicitor involving cerebroside A, B, or C was detected in other soilborne phytopathogens, such as Pythium and Botrytis. Treatment of lettuce (Lactuca sativa), tomato (Lycopersicon esculentum), melon (Cucumis melo), and sweet potato (Ipomoea batatas) with cerebroside B resulted in resistance to infection by each pathogenic strain of F. oxysporum. Induction of pathogenesis-related genes and H(2)O(2) production by treatment with cerebroside B were observed in tomato root tissues. The cerebroside elicitor showed no antifungal activity against F. oxysporum in vitro, indicating that the cerebroside elicitor activates defense mechanisms to confer resistance to Fusarium disease. These results suggest that cerebroside functions as a non-race-specific elicitor in a wide range of plant-phytopathogenic fungus interactions. Additionally, cerebroside elicitor serves as a potential biologically derived control agent.     

Laser-stimulated luminescence used to measure x-ray diffraction of a contracting striated muscle

An integrating x-ray area detector that operates on the basis of laser-stimulated luminescence was used in a diffraction study of muscle contraction. The area detector has a dynamic range of 1 to 10(5), a sensitivity about 60 times greater with approximately 1/300 as much fog background as x-ray film. It is erasable and reusable but, like film, can integrate at a practically unlimited counting rate. The high sensitivity and wide dynamic range of the detector resulted in a sufficient reduction in the exposure time to make possible the recording of a clear x-ray diffraction pattern, with up to 2.0-nanometer axial spacing, from a contracting frog skeletal muscle in as little as 10 seconds with synchrotron radiation. During the isometric contraction of the muscle, most of the actin diffraction lines increased in intensity without noticeable changes in their peak positions. Changes also occurred in diffraction intensities from the myosin heads. The results indicate that during contraction the structure of the actin filaments differs from that in the rigor state, suggesting a possible structural change in the actin subunits themselves; the myosin heads during contraction retain the axial periodicity of the myosin filament and become aligned in a more perpendicular manner to the actin filaments.     

Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.     

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)₅, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.